The CCAAT box is a prototypical promoter element,almost invariably found between 60 and 100 upstreamof the major transcription start site. It is bound and acti-vated by the histone fold trimer NF-Y. We performed chro-matin immunoprecipitation (ChIP) on chip experimentson two different CpG islands arrays using chromatin fromhepatic HepG2 and pre-B cell leukemia NALM-6 cell lines,with different protocols of probe preparation and label-ing. We analyzed and classified 239 known or predictedtargets; we validated several by conventional ChIPs withanti-YB and anti-YC antibodies, in vitro EMSAs, and ChIPscanning. The importance of NF-Y binding for gene ex-pression was verified by the use of a dominant negativeNF-YA mutant. All but four genes are new NF-Y targets,falling into different functional categories. This analysisreinforces the notion that NF-Y is an important regulatorof cell growth, and novel unexpected findings emergedfrom this unbiased approach. (i) A remarkable proportionof NF-Y targets, 40%, are complex transcriptional unitscomposed of divergent, convergent, and tandem promot-ers. (ii) 4050% of NF-Y sites are not in core promoters butare in introns or at distant 3 or 5 locations. The abun-dance of unorthodox CCAAT positions highlights an un-expected complexity of the NF-Y-mediated transcrip-tional network.

The CCAAT box is a DNA element that controls transcrip-tional initiation in eukaryotic promoters; recent bioinformaticstudies unambiguously identify it as one of the most wide-spread. The analysis on 1031 human promoters isolatedthrough unbiased determination of mRNA start sites suggestedthat the CCAAT box or its reverse ATTGG is present in asmany as 67% of promoters (1). A statistical, unbiased analysisof random octanucleotides on a large 13,000-promoter data set

confirmed that the CCAAT is second only to the Sp1-bindingGC box in terms of abundance, despite the fact that the per-centage of CCAAT promoters was inferior, 7.5% (2). Further-more, analysis of cell cycle-regulated genes identified theCCAAT box as specifically present in promoters of G2/M genes(3). Most importantly, specific flanking nucleotides emergingfrom these studies matched specifically the consensus of theNF-Y transcription factor. A combination of EMSAs and trans-fections with highly diagnostic dominant negative vectors im-plicated NF-Y as the CCAAT activator (4). It is composed ofthree subunits, NF-YA, NF-YB, and NF-YC, all necessary forsequence-specific binding to a G/A, G/A, C, C, A, A, T, C/G, A/G,G/C consensus. NF-YB and NF-YC contain evolutionarily con-served histone fold motifs common to all core histones, medi-ating dimerization, a feature strictly required for NF-YA asso-ciation and sequence-specific DNA binding (5, 6). In essentiallyall cases described so far, the binding of the trimer is importantor essential for transcriptional regulation (7).

NF-Y is considered as a general promoter organizer: thanksto its histone-like nature, it presets chromatin structure locally(8), interfacing well with nucleosomes (9), it helps the bindingof neighboring factors (reviewed in Refs. 4 and 5) and attractscoactivators, such as p300/CREB-binding protein (8, 10). Thelocation of the CCAAT box is far from random, being positionedbetween 60 and 100 in the vast majority of the promotersanalyzed. In general, our knowledge of the anatomy of NF-Y-binding sites in terms of flanking sequences, position withrespect to transcriptional start sites, and promoter context (6,11, 12) enables us to make predictions as to whether a gene willbe regulated by NF-Y.

Chromatin Immunoprecipitation (ChIP)1 experiments deter-mined that NF-Y is bound in vivo before gene activation (1013); NF-Y is bound to a transcribing cyclin B1 promoter duringmitosis in HeLa cells (14). Indeed, binding to cell cycle-regu-lated promoters is not constitutive but is time-regulated, beingfound before activation and displaced when promoters are re-pressed (10). Furthermore, conditional knock-out experimentsof CBF-B (NF-YA) unambiguously determined that the proteinis required for cell proliferation of mouse embryo fibroblastsand mouse development (15).

The analysis of 130 mammalian CCAAT-containing promot-ers suggests a prevalence in genes that are active in a tissue- or

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 14, Issue of April 8, pp. 1360613615, 2005 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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development-specific way and in inducible genes, either byexternal stimuli or during the cell cycle (7). Whereas this iscertainly informative, very little information exists as to thebinding to other regions. Finding all genes targeted by a par-ticular transcription factor is crucial to reconstruct its tran-scriptional network. To expand our knowledge of NF-Y bindingin vivo, a valuable approach is to use DNA derived from ChIPsto probe microarrays. DNA arrays have been developed inwhich clones derived from a CpG island library have beenspotted (16); CpG islands have long been known to be associ-ated to regulatory elements in promoters (17) and also else-where in the genome. They are believed to be mainly associatedto housekeeping genes (i.e. genes active in all cells), albeit atdifferent levels (reviewed in Ref. 18). To gain a wider under-standing of the NF-Y transcriptional circuitry, we took a highthroughput genomic approach by screening with anti-YB chro-matin-immunoprecipitated DNA two CpG island arrays.

Data validation was performed with conventional ChIPs (10), withchromatin of 0.8 kb and with anti-YB as well as anti-YC purifiedpolyclonal antibodies. The sequence of PCR primers used to analyze thegenes reported in Fig. 2 are shown in Supplemental Table I.

Generation of ChIP Probes

DNAs from 2030 individual ChIPs were used to generate a probe forarray screening. Immunoprecipitated chromatin was used as templatefor random priming reactions in the presence of 10 mM amino allyl-UTP(Sigma catalog no. A-0410) using the BioPrime DNA labeling system(Invitrogen). The DNAs were desalted and concentrated with a Micro-con YM30 filter column (Millipore Corp.) and then lyophilized. Afterresuspension in water, amino allyl-dUTP-labeled chromatin was cou-pled with Cy5 dye (Amersham Biosciences) solubilized in 0.1 M sodium

bicarbonate, pH 9.0, for 1 h in the dark. After the addition of 0.1 Msodium acetate, pH 5.2, DNAs were purified with QIAquick columns(Qiagen) and lyophilized.

Amplicon Generation and Labeling

The generation of amplicons from individual ChIPs was performedfollowing the protocols of LM-PCR described in Refs. 20 and 21. Briefly,two unidirectional linkers (oligonucleotide JW102, 5-GCGGTGAC-CCGGGAGATCTGAATTC-3; oligonucleotide JW103, 5-GAATTCA-GATC-3) were annealed and ligated to the chromatin IPs, previouslyblunted by T4 DNA polymerase. The first amplicons were generated byPCR (one cycle at 55 C for 2 min, 72 C for 5 min, 95 C for 2 min,followed by 15 cycles at 95 C for 30 min, 55 C for min, 72 C for 1 min,and a final extension of 4 min at 72 C). The reaction was purified usingthe Qiaquick PCR purification kit (Qiagen) or the GFX PCR purificationkit (Amersham Biosciences) according to the manufacturers instruc-tions. One-tenth of these initial reactions were used to generate moreamplicons, using the same PCR program for a subsequent 30 cycles.After purification of these last rounds of amplification, the DNA wasquantified and examined by gene-specific PCR to ensure that the initialenrichment was maintained. 5 g of amplicons for -NF-YB, -FLAG,and input DNA (subjected to the same number of PCR manipulations asthe IPs) were labeled using the LabelIT Cy5/Cy3 nucleic acid labelingkit (Mirus), following the manufacturers instructions, with a reagent/DNA ratio of 2.5 for Cy5 (IPs) and 1.5 for Cy3 (input).

CpG Microarray Hybridization

7776 CpG ArrayThe development of the 7776 CpG island arraywas described previously (2123). Prior to hybridization, spotted CpGisland slides were incubated with a solution of 3 SSC, 0.25% SDS, and1.5 g/l salmon testis DNA under a glass coverslip at 37 C for 30 minto block nonspecific binding. Slides were washed twice with water anddried for 5 min at 600 rpm in a centrifuge. Labeled DNAs were added tohybridization buffer (0.25 M NaPO4, 4.5% SDS, 1 mM EDTA, and 1SSC), denatured at 95 C for 2 min, cooled to 60 C, and dropped ontoslides placed in prewarmed hybridization chambers. Incubation wasperformed at 60 C overnight. After hybridization, the slides werewashed successively at 50 C with 1 SSC, 0.1% SDS at room temper-ature with 1 SSC (0.1%) and at room temperature with 0.2 SSC for 5min each and then dried. Hybridized slides were scanned with theGenePix 4000A scanner (Axon), and the acquired images were analyzedwith the software GenePix Pro, Version 3.0. A global normalizationfactor was determined for each replica, evaluating the anti-NF-YBChIP Cy5/control ChIP Cy5 ratio relative to control repetitive elements.Data were normalized prior to comparison. After normalization, posi-tive loci were defined by hybridization intensities at least 2 timesgreater than that of control.

12K ArrayThe Cy5- and Cy3-labeled DNA were each resuspendedin 10 l of 1 g/l Cot-1 DNA (Invitrogen) and mixed together in orderto have the same amount of input Cy3-labeled DNA for each IP Cy5-labeled DNA. The hybridization solution was then added to a finalcomposition of 43% formamide, 4.3 SSPE, 0.42% SDS, 42 g of salmonsperm DNA, 0.2 g of tRNA, heated for 2 min at 95 C and cooled downto 37 C over 30 min. 95 l of each mixture solution was applied to twohuman CpG 12K slides (University Health Network, The MicroarrayCenter, Toronto, Canada) and hybridized at 37 C for 18 h. The slideswere prehybridized for 1 h at 42 C with 25% formamide, 5 SSC, 0.1%SDS, and 10 g/l bovine serum albumin.

The slides were washed at room temperature for 5 min twice in 2SSC, 0.1% SDS; once in 1 SSC, 0.1% SDS; and one final time in 0.1SSC; dried; and immediately scanned using a ScanArray 4000 scanner(Packard). The hybridized microarrays were an...